The long term goals of this project are to identify gene regulatory mechanisms involved in expression of the osteoblast phenotype using a well defined cell culture system. The proposed work will clone and characterize mRNA sequences that are preferentially expressed in differentiating osteoblasts. These clones will be used to study early changes in gene expression that accompany induction of the osteoblast phenotype. They can also be used to identify osteoblast precursors in different stages of maturation. Specific Aims are: 1) to clone mRNA sequences selectively expressed at early times during the in vitro differentiation of MC3T3-El preosteoblast cells and, 2) to characterize early-induced mRNA sequences. Cloning will be accomplished using subtractive cDNA hybridization to produce populations enriched in mRNA sequences from differentiating or control cells. The developmental expression and nucleotide sequence of these mRNAs will be determined. Isolation of these clones will serve as the basis for a major funding application to fully characterize induced sequences and determine their role in osteoblast differentiation. This project will increase our understanding of factors regulating osteogenesis, knowledge which is necessary for the eventual treatment of degenerative bone disorders such as periodontal disease, osteoporosis, and non-union fractures.